Arnold J. Smolen

Neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods for cell counting

SummaryPublished values for the number of neurons in the superior cervical ganglion of the adult rat range from 13 000 to 45 000. These studies have employed different methods for determining what unit to count (cell body, nucleus, nucleolus), how many sections to count, and how to correct the raw counts for split particles and for profiles that are too small to resolve. The purpose of this study was to examine the extent to which these parameters may influence the calculated value for the total number of neurons, using computer simulations of neuron populations. These simulations permitted us to determine the effects on neuron number of varying the diameter of the neuronal nucleus, the size of the smallest resolvable profile, and the thickness of the section. The data from the simulations were used to test the validity of several methods that are in common use for correction of neuron counts. Our results indicate that most of the methods that are in routine use are unsatisfactory. We propose the use of either one of two methods that consistently result in highly accurate estimates of neuron numbers. These are: (1) a modification of the method proposed by Hendry (1976), using computer analysis; or (2) a modification of the method proposed by Abercrombie (1946), which does not require the use of a computer.

Developmental neuron death in the rat superior cervical sympathetic ganglion: cell counts and ultrastructure

SummaryCounts of neurons of the rat superior cervical ganglion (SCG) were made at two days before birth and at several postnatal ages. There is a significant decline in the number of apparently normal neurons over the first postnatal week, with the number falling from 39 500 at 3 days to 26 500 at 7 days. Cell numbers then remained constant up to day 60 when the number of neurons was 27 500. The incidence of degenerating neurons, identified by light and electron microscopy, was correlated temporally with the loss of normal neurons. The early manifestations of the neuron degeneration were chromatin clumping and the presence of free monoribosomes. Later stages were characterized by increased chromatin clumping, dense aggregations of monoribosomes, numerous intracytoplasmic vacuoles, and only short segments of rough endoplasmic reticulum. The ultrastructure of the majority of these dying neurons is similar to the ‘nuclear’ types of degeneration described by Pilar & Landmesser (1976) and Chu-Wang & Oppenheim (1978). Based on the presence of degenerating neurons coincident with the reduction in neuron numbers, we conclude that neuron death is an important aspect of early postnatal development in the rat SCG.

Neonatal testosterone treatment increases neuron and synapse numbers in male rat superior cervical ganglion

Neonatal treatment with gonadal steroids has been reported to alter morphological as well as functional development in various regions of the brain and spinal cord. Among the observed alterations are changes in numbers of neurons and in the organization and numbers of synapses. These regions have been found to be sexually dimorphic, and the dimorphism dependent upon gender differences in circulating levels of gonadal steroids. Neonatal treatment with testosterone has been shown to produce an increase in the number of neurons in the superior cervical sympathetic ganglion in female rats. The present studies were designed to investigate the possibility of a normally occurring sexual dimorphism in the SCG of the rat, and to characterize the effect of neonatal treatment with testosterone on neurons and synapses in the male rat. We report a sexual dimorphism in the number of neurons but not in the number of preganglionic axons or ganglionic synapses. In addition, neonatal administration of testosterone propionate results in a 40% increase in the number of superior cervical ganglion neurons in treated male rats over the control male number at 15 and 30 days of age. The testosterone propionate treatment results in a 66% increase in the number of synapses in male superior cervical ganglia, without a concomitant increase in the number of preganglionic axons.

Retrograde transneuronal regulation of the afferent innervation to the rat superior cervical sympathetic ganglion.

SummaryThe superior cervical sympathetic ganglion of the rat receives its preganglionic afferent innervation through the cervical sympathetic trunk, and sends most of its postganglionic axons through two major nerves, the internal (ICN) and external carotid nerves (ECN). In the present study, the ICN alone or both the ICN and the ECN were cut in neonatal and adult rats. Two months after these lesions, ganglionic neurons, synapses and preganglionic axons were counted and compared with unoperated control values. After cutting the ICN alone in neonatal rats, ganglionic neurons were reduced in number by 70% and synapses were reduced by 50%, but there was no change in the number of preganglionic axons. Cutting both the ICN and the ECN in neonates resulted in an 88% reduction of ganglionic neurons and an 83% reduction of synapses. In this case there was a 63% reduction in the number of preganglionic axons. After cutting either the ICN alone or both the ICN and the ECN in neonates, there was a hyperinnervation (increased number of synapses) of the remaining ganglionic neurons. In the adult rat, cutting either the ICN alone or both the ICN and ECN resulted in a smaller loss of ganglionic neurons, and there was no loss of preganglionic axons. There was no hyperinnervation of surviving neurons in adult rats. Thus, the response by preganglionic axons to a reduced number of ganglionic neurons differs in the neonate and adult rat. In the developing animal, the degenerative response to injury is much more severe than in the adult, but the reorganizational response is also greater.

Effects of 17-β-estradiol on developing superior cervical ganglion neurons and synapses

Abstract Previous studies have shown that neonatal treatment of male or female rats with testosterone propionate dramatically increases the number of neurons and synapses in the superior cervical sympathetic ganglion. We now report that administration of another gonadal steroid, 17-β-estradiol, over the first 2 postnatal weeks nearly doubles the number of postganglionic neurons and synapses in the male superior cervical ganglion.

Postnatal development of ganglionic neurons in the absence of preganglionic input: Morphological observations on synapse formation

Synapse formation during postnatal development of the superior cervical sympathetic ganglion was studied in rats after neonatal ganglionic denervation, when reinnervation was either permitted or prevented. In both groups of operated animals, 90% of the synapses were lost by the fourth postoperative day. In the rats in which reinnervation was permitted, restoration of synapse numbers began by one month after surgery and reached 50% of control by two months. There was no further synapse restoration after this time. In the animals in which reinnervation was prevented, synapse numbers increased, but at all times were approximately 10% of controls. Thus the intrinsic ganglionic synapses underwent their normal postnatal developmental increase in number, but did not sprout to any significant degree in response to the massive deafferentation caused by removal of the preganglionic input. Vacant postsynaptic membrane thickenings (those not apposed by presynaptic terminals) appeared in both experimental groups. There was no significant loss of these vacant thickenings in either group over the course of 3 months. Therefore, the vacated postsynaptic sites do not appear to be recontacted during reinnervation, while the reinnervating axons appear to cause the formation of new postsynaptic sites on the ganglion cells.

Effects of neonatal castration or treatment with dihydrotestosterone on numbers of neurons in the rat superior cervical sympathetic ganglion

There is a normal sexual dimorphism in the number of neurons in the rat superior cervical sympathetic ganglion (SCG), with adult males having more neurons in this ganglion than females. We confirm this finding here, report that this sex difference is not present at birth and that neonatal castration of males reduces the adult sex difference. These results indicate that perinatal levels of circulating testicular hormones play a role in regulating the numbers of neurons in the SCG. Treatment of neonatal rats with testosterone propionate or estradiol significantly increases the number of neurons in the SCG. To determine whether this effect is primary androgenic or estrogenic, the effects of dihydrotestosterone, on SCG neuron number were investigated. Dihydrotestosterone, unlike testosterone, is not aromatized to estradiol intracellularly. There was no difference in the number of neurons between animals injected from birth with vehicle or dihydrotestosterone. This difference in effects between dihydrotestosterone and testosterone suggests that the actions of testosterone may be via aromatization to estradiol, rather than action at an androgen receptor.

Neurotransmitter synthesis, storage, and turnover in neonatally deafferented sympathetic neurons.

In the superior cervical sympathetic ganglion (SCG) of the rat, a significant amount of morphological and biochemical maturation occurs in the first few postnatal weeks. The specific activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine (NE), increases during this time and is subject to transsynaptic regulation by the preganglionic inputs. In the present study, we examined the normal postnatal development of NE stores in sympathetic neurons and the transsynaptic regulation of this development. NE content undergoes an 8-fold increase from the time of birth, and stabilizes at adult levels at one month. Following neonatal deafferentation, there is a temporary stunting of NE accumulation in sympathetic neurons and a permanent reduction in the activity of TH, whether or not regeneration of the afferents occurs. When regeneration is prevented, the turnover of NE is significantly reduced, while NE levels rise to near normal levels. When regeneration is permitted, however, both the stored amount and turnover of NE attain normal levels. These data suggest that there is a critical period during the first two postnatal weeks when transsynaptic influences from afferents are necessary for the induction of TH in sympathetic neurons. Levels and turnover of transmitter do not have this critical period, but appear to depend solely on the functional integrity of the system.

Gender differences in neurotransmitter expression in the rat superior cervical ganglion

Sexual dimorphism of neuron number has been observed in several areas in the central and peripheral nervous system. In many of these areas enhanced neuron survival exists in males during the period of naturally occurring cell death. This has been attributed to high levels of circulating testosterone in the perinatal period. The superior cervical ganglion (SCG) of the rat exhibits this sexual dimorphism. The difference in neuron numbers is established by two weeks postnatally and precedes the differences in body weight and sympathetic target mass between the sexes. At this two week time point, fewer SCG neurons in the female rat must supply neurotransmitter to the same mass of sympathetic target as in the male. The present study examined some of the mechanisms used by neurons in the SCGs of male and female rats to compensate in supplying neurotransmitter to their targets. At birth, the SCGs of male and female rats contain equal numbers of neurons. There is also no sex difference at this time in the content of norepinephrine (NE) in these neurons or in the enzyme activity of tyrosine hydroxylase (TH). However, a sex difference does exist in the expression of TH-mRNA, with SCG neurons in female expressing more TH-mRNA than males. At this time, there is no sex difference in either the total body weight of males and females or in the mass of sympathetic target organs. During the first two postnatal weeks, natural neuron death in the SCG results in the loss of significantly more neurons in females than in males. At the end of the period of cell death, neurons in females continue to express more TH-mRNA, and at this time both TH enzyme activity and NE content per neuron are also higher in females. Since the adult sex difference in body weight and sympathetic target mass has not yet been established, the same amount of target mass is innervated by fewer neurons in females. In the adult, the sex difference in SCG neuron number is maintained. However, both overall body weight and sympathetic target mass are significantly greater in males. At this time expression of TH-mRNA is greater in SCG neurons of males, while both TH enzyme activity and NE content per neuron are equal in males and females. One of the challenges presented to the developing nervous system is to match a population of neurons with its targets.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptogenic effects of neonatal estradiol treatment in rat superior cervical ganglia

Neonatal rats treated with testosterone propionate or 17-beta-estradiol during the first two postnatal weeks have more neurons and synapses in their superior cervical ganglia (SCGs) at 15 days of age than do vehicle-treated littermates. To determine whether a non-aromatizable androgen would similarly increase the number of SCG synapses, dihydrotestosterone (DHT) was injected into male rats beginning on the day of birth. The animals were sacrificed on postnatal day 15 and the SCGs removed on postnatal day 15. Counts of synapses showed no difference in the number of synapses between control and DHT-treated animals. These results suggest that the actions of testosterone to increase the numbers of SCG synapses may be via aromatization to estradiol. An additional study was done to determine whether the additional synapses formed in SCGs of animals treated with estradiol arise from neurons whose axons are in the cervical sympathetic trunk or from intrinsic neurons, i.e., SIF cells or other principal ganglion neurons. Neonatal males were injected with 17-beta-estradiol or vehicle beginning on the day of birth and continuing until the time of sacrifice on day 15. The number of intrinsic synapses formed under control and estradiol treatments was determined in SCGs of animals whose extrinsic synapses were caused to degenerate by severing the cervical sympathetic trunk bilaterally on postnatal day 13, two days before sacrifice. The total number of synapses (extrinsic plus intrinsic) in the ganglion after vehicle or estradiol treatment was determined in unoperated animals and used to calculate the number of extrinsic synapses.(ABSTRACT TRUNCATED AT 250 WORDS)